Oral killed vaccines and method for providing same

ABSTRACT

There is described a method for selecting microbial isolates for use in oral killed vaccines against abnormal microbial colonization of mucosal surfaces by the microbes. The method comprises evaluating capacity of a plurality of different isolates of a microbe to activate antigen responsive cells to provide activation data for each microbial isolate, and the effectiveness of the isolates in reducing infection of a mucosal surface by the microbe to provide clearance data for each microbial isolate. An isolate, the activation data and clearance data for which correlate and is optimal for generating mucosal immunity against the microbe compared to the, or each, other of the isolates, or an isolate the activation data for which is optimal and a further isolate the clearance data for which is optimal, compared to the, or each, other of the isolates, respectively, is then selected for use in the vaccine. There is also described a method for providing an oral killed vaccine against abnormal microbial colonization of a mucosal surface, comprising evaluating the capacity of a plurality of different isolates of a microbe to induce expression of IL-10 and IL-12 in antigen responsive cells. At least one isolate is selected that induces optimal expression of IL-12 relative to IL-10 compared to the, or each, other of the isolates, respectively, for use in the vaccine.

FIELD OF THE INVENTION

The present invention relates to a method for selecting unicellularmicrobial isolate(s) for use in oral killed vaccines, for inducing aprotective mucosal immune response against abnormal colonization of amucosal surface by the microbe. Oral killed vaccines for the prophylaxisor treatment of such infections are also provided.

BACKGROUND OF THE INVENTION

Anti-bacterial vaccines are known in the art and examples includeHaemophilus influenzae B vaccine which consists of bacterialpolysaccharide conjugated with tetanus toxoid protein. Killed bacterialvaccines for the prophylaxis or treatment of enteric infections havealso been known for some time and a killed bacterial vaccine for typhoidfever is commercially available. These vaccines are predominantly if notexclusively administered by injection and serve as “classic” vaccines inthat they aim to stimulate a systemic antibody response to provideprotection against disease.

Antigen administered orally is processed by gut-associated lymphoidtissue (GALT) differently from systemic lymphoid tissue. Teleologically,this can be understood in terms of mucosal physiologically whereenvironmental “antigen” needs to be excluded but not at the cost ofdamaging mucosal “inflammation”. A powerful suppression mechanismtherefore exists, to minimize potentially damaging immune responses tosuch antigen. This concept was originally identified as “splittolerance” where a systemic immune response (ie. mediated by thegeneration of antibody) was associated with the failure to detect amucosal antibody response (tolerance). Research using orallyadministered killed influenza virus shows that an antibody response isstimulated over a narrow range of antigen dose. This immunization “zone”is flanked by low and high “zone” tolerance. The same concept applies tocellular immunity though the zone in which T-lymphocyte-mediatedresponses may be stimulated appears to be marginally wider withprotection occurring without an antibody response. The outcome ofantigen interaction with GALT is the selective migration of B andT-lymphocytes to distant mucosal sites of infection where they mediateprotection.

An oral killed bacterial vaccine against infection by non-typeableHaemophilus influenzae (NTHi) is also known in the art. NTHi is thebacteria most commonly linked with nasal and bronchus colonization insubjects with chronic lung disease, and has been linked to acuteepisodes of bronchitis in these subjects. A significant factor in thegeneration of acute bronchitis in such subjects is the uncontrolled andinappropriate migration of neutrophils into the bronchus lumen inresponse to the colonizing bacteria. The accumulation ofneutrophil-laden fluid within the bronchi results in purulent sputum.The use of the oral NTHi killed bacterial vaccine has been shown toprotect against purulent sputum production, high levels of bacterialcolonization of the airways and environmental spread of the bacteria asassessed by acquisition of infection by bystander subjects. The NTHivaccine stimulates the common mucosal system following activation ofGALT and more specifically, Peyer's patches in the intestines.

Oral non-adjuvenated monobacterial vaccines comprising killed bacteriafor providing mucosal immunity particularly in patients with long termchronic lung disease are described in international patent applicationNo. PCT/AU86/00071. Specifically, the application indicates that theimmunization efficacy of the vaccine arises from the absence ofadjuvant, which would normally be included in such vaccine formulationsto promote an immune response. The generation of the immune response inthe absence of the adjuvant was reasoned to be due to the responseelicited by the killed bacteria being insufficient to trigger dominantsuppressor T-cell populations in the lungs of the patients evaluated.

SUMMARY OF THE INVENTION

Broadly stated, the present invention relates to the provision of oralkilled vaccines for protecting against abnormal microbial colonizationand stems from the recognition that there is a marked variation in theclearance of such infections elicited by oral killed vaccines in anoutbred population, reflecting the genetic variation in the population.The variation in mucosal immunity associated with the use of prior artoral killed bacterial vaccines is believed to arise from the use of lessoptimal or randomly chosen microbial isolates in the vaccines, due tothe failure to recognize the significant variability in the capacity ofdifferent isolates of a microbe to activate antigen-presenting cells andT-lymphocytes. Given the observed variability, the selection of theisolate(s) is critical for optimizing the degree of activation of thecommon mucosal system in different individuals in an outbred population.Methodology provided in one or more embodiments of the present inventionenables the selection of isolate(s) for optimizing oral killed vaccines.

More particularly, in one aspect of the present invention there isprovided a method for selecting a microbial isolate for an oral killedvaccine against abnormal microbial colonization of a mucosal surface,the method comprising:

-   -   evaluating capacity of a plurality of different isolates of a        unicellular microbe to activate antigen responsive cells to        provide activation data for each microbial isolate;    -   evaluating effectiveness of the isolates in reducing infection        of a mucosal surface by the microbe to provide clearance data        for each microbial isolate; and    -   selecting an isolate from the microbial isolates, the activation        data and clearance data for which correlate and is optimal for        generating mucosal immunity against the microbe compared to the,        or each, other of the isolates, or an isolate the activation        data for which is optimal and a further isolate the clearance        data for which is optimal, compared to the, or each, other of        the isolates, respectively, for use in formulating the vaccine.

In another aspect of the present invention there is a method forproviding an oral killed vaccine against abnormal microbial colonizationof a mucosal surface, the method comprising:

-   -   evaluating capacity of a plurality of different isolates of a        unicellular microbe to activate antigen responsive cells to        provide activation data for each microbial isolate;    -   evaluating effectiveness of the isolates in reducing infection        of a mucosal surface by the microbe to provide clearance data        for each microbial isolate;    -   selecting an isolate from the plurality of isolates, the        activation data and clearance data for which correlate and is        optimal for generating mucosal immunity against the microbe        compared to the, or each, other of the isolates, or an isolate        the activation data for which is optimal and a further isolate        the clearance data for which is optimal, compared to the, or        each, other of the microbial isolates, respectively, for use in        formulating the vaccine; and    -   formulating the vaccine using the selected isolate or isolates.

Typically, an isolate for which both the activation data and theclearance data is optimal compared to the other of the isolates will beselected.

Preferably, the mucosal immunity will comprise predominantly a cellularimmune response.

Preferably, the isolates utilized for providing the activation andclearance data will be killed isolates of the microbe. However, theinvention is not limited thereto and the activation and clearance datamay be obtained from live isolates and the selected isolate(s)subsequently killed for use in the vaccine.

The antigen responsive cells activated by the isolate(s) will normallycomprise one or both of antigen presenting cells and T-lymphocytes andpreferably, will comprise both types of cells. The antigen presentingcells will typically comprise macrophages. Most preferably, theT-lymphocytes will be Th1 cells. The activation of the antigenresponsive cells is to be taken in its broadest sense to encompassdirect and/or indirect activation by the isolate(s). By “direct”activation is meant the isolate(s) activate at least some of the antigenresponsive cells by contact with the cells such as when a microbialisolate is bound or phagocytosed by them. By “indirect” activation ismeant at least some of the antigen responsive cells are activated byinteraction with the cells such as macrophages that have contacted theisolate or for instance, by cytokine(s) or other chemical messenger(s)the release of which has been elicited or induced by the isolate(s), orby substances such as toxins or antigens secreted by the isolate(s), ora combination of the foregoing possibilities.

The level of activation of the antigen responsive cells can be evaluatedby measuring one or more parameters associated with activation of thecells. Preferably, the capacity of the isolate(s) to activate bothantigen presenting cells and T-lymphocytes will be evaluated. Inparticularly preferred embodiments, the activation of the antigenresponsive cells achieved by each isolate will be evaluated by measuringat least one parameter indicative of the level of activation of theantigen presenting cells and at least one other parameter indicative ofthe level of activation of the T-lymphocytes. Most preferably,isolate(s) the activation data for which is optimal for activating bothantigen presenting cells and T-lymphocytes compared to the otherisolate(s) tested will be selected for use in the preparation of theoral killed vaccine.

Preferably, an isolate the activation data for which is indicative ofthe capacity of the isolate to elicit a cytokine response characterizedby an IL-10:IL-12 ratio of about 30 or less will be selected for use informulating the oral killed vaccine.

Hence, in another aspect of the present invention there is provided amethod for selecting a microbial isolate for an oral killed vaccineagainst abnormal microbial colonization of a mucosal surface, the methodcomprising:

-   -   evaluating capacity of a plurality of different isolates of a        unicellular microbe to induce expression of IL-10 and IL-12 in        antigen responsive cells; and    -   selecting at least one isolate from the microbial isolates, that        induces optimal expression of IL-12 relative to IL-10 compared        to the, or each, other of the isolates, respectively, for use in        formulating the vaccine.

In still another aspect of the present invention there is provided amethod for providing an oral killed vaccine against abnormal microbialcolonization of a mucosal surface, the method comprising:

-   -   evaluating capacity of a plurality of different isolates of a        unicellular microbe to induce expression of IL-10 and IL-12 in        antigen responsive cells;    -   selecting at least one isolate from the microbial isolates, that        induces optimal expression of IL-12 relative to IL-10 compared        to the, or each, other of the isolates, respectively, for use in        formulating the vaccine; and    -   formulating the vaccine using the isolate.

The unicellular microbe can be any such microbe having the capacity tocolonize a mucosal surface of a mammal and may for instance be selectedfrom the group consisting of bacteria, fungi and yeast. Typically, themicrobe will be a bacteria and the vaccine will therefore be an oralkilled bacterial vaccine. Preferably, the selected isolate(s) will beused in the vaccines of the invention as whole killed organisms.However, the invention is not limited to the use of whole killedorganisms and vaccines may be provided comprising particulate matterderived from the outer cellular membrane of the selected isolate(s).

In yet another aspect, the present invention relates to an oral killedvaccine provided by a method of the invention.

A vaccine of the invention may be directed against infection of anymucosal site including chronic and acute such infections. The infectioncan be the result of transient exposure to a microbial pathogen whichdoes not normally colonise the mucosal site or for instance, anopportunistic infection arising from microbial flora normally found atthe site.

Accordingly, in another aspect, there is provided a method for theprophylaxis or treatment of an infection of a mucosal surface in amammal by a unicellular microbe, the method comprising:

-   -   administering to the mammal an effective amount of an oral        killed vaccine of the invention for generating mucosal immunity        against the microbe.

The mammal may be any mammal treatable with an oral killed bacterialvaccine of the invention. For instance, the mammal may be a primate, amember of the rodent family such as a rat or mouse, or a member of thebovine, porcine, ovine or equine families. Preferable, however, themammal will be a human being.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

All publications mentioned in this specification are herein incorporatedby reference. Any discussion of documents, acts, materials, devices,articles or the like which has been included in the presentspecification is solely for the purpose of providing a context for thepresent invention. It is not to be taken as an admission that any or allof these matters form part of the prior art base or were common generalknowledge in the field relevant to the present invention as it existedin Australia or elsewhere before the priority date of each claim of thisapplication.

The features and advantages of the present invention will become furtherapparent from the following description of preferred embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing reduction of NTHi colonization in therespiratory tract of BALB/c mice by different whole cell killed isolatesof the bacteria administered orally.

FIG. 2 is a graph showing reduction of S. aureus colonization in thenose of BALB/c mice by different whole cell killed isolates of thebacteria administered orally.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Vaccines embodied by the invention find particular application in theprophylaxis or treatment of lung and upper respiratory tract infections.However, the invention is not limited thereto and mucosal immunityresulting from activation of the common mucosal system may provideprotection or treatment against infections at other mucosal sites of thebody including infections of the oral, nasal, oropharyngeal, nasalpharyngeal, pharyngeal, digestive, vaginal, eye associated, and urinarymucosal surfaces. The vaccine may contain bacteria selected for instancefrom Chlamydia species, Haemophilus species, Non-typeable Haemophilusspecies, Pseudomonas species, Streptococcus species, Staphylococcusspecies, E. coli species, Mycoplasma species and Helicobacter speciesamongst others, or incorporate combinations of different species ofbacteria or of other unicellular microbes. Microbes other than bacteriathat may be used in oral killed vaccines according to the inventioninclude Candida species such as Candida albicans and yeast species suchas Saccharomyces species. Particularly preferred oral killed bacterialvaccines embodied by the invention are vaccines for the prophylaxis ortreatment of mucosal infections by bacteria selected from the groupconsisting of Non-typeable H. influenzae (NTHi), S. aureus, P.aeruginosa, S. pneumoniae and combinations thereof.

While the primary application of a vaccine embodied by the invention isto generate mucosal immunity against the particular bacterialinfection(s) for which the vaccine is provided, which may occur atvarious mucosal sites, the vaccine can also be used for the treatment orprophylaxis of diseases or conditions exacerbated by the infection(s).

P. aeruginosa for instance can colonise not only the respiratory tractbut can also infect eye mucosa and the ear cavity. Non-typeable H.influenzae (NTHi) has also been implicated in a range of infectiousconditions including otitis media and in the exacerbation of pneumoniaand chronic bronchitis. Accordingly, a vaccine containing one or morekilled NTHi isolates of this bacteria may be administered for theprophylaxis or treatment of those conditions. Similarly, vaccines of theinvention comprising killed H. influenzae, S. pneumoniae or P.aeruginosa may be utilized in the prophylaxis or treatment of bronchitisor pneumonia, and acute infections in cystic fibrosis and chronicobstructive airways disease, sinus disease, compromised lung functionand other lung and respiratory tract diseases and disorders. Thesevaccines also find particular application in the prophylaxis ortreatment of superinfection by the corresponding bacteria followinginfection by influenzae virus or other virus, particularly in theelderly.

While it is preferable to use whole killed isolate(s) in vaccines of theinvention, particulate cell surface matter comprising surface antigensof the isolate(s) may be utilized as well, or instead of, whole killedorganisms. In a particularly preferred embodiment, the outer cellularmembrane fraction of the organisms will be utilized. The particulatematter can be prepared by disrupting killed or viable selectedisolate(s) by sonication or other suitable technique and if desired,separating the required fraction from other cellular components such asby centrifugation, filtration and/or other appropriate technique knownin the art. Any suitable method which achieves the required level ofcellular disruption may be employed including sonication or dissolutionutilizng appropriate surfactants and agitation. When sonication isemployed, the isolate may be subjected to a number of sonication stepsin order to obtain the desired degree of cellular disruption orgeneration of particulate matter of a desired size. The fraction ofparticulate matter utilized may be selected by comparing the response ofantigen responsive cells to different fractions of the isolate(s) andselecting the fraction which maximizes the immune response by the cells.

To evaluate the capacity of a microbial isolate or particulate matterthereof to activate the antigen responsive cells, any parameter which isindicative of the level of activation of the cells may be evaluated.Particularly, preferred parameters include one or more of cellularproliferation, cell surface antigen expression, measurement of celleffector functions, and cytokine production.

Cellular proliferation and in particular, T-cell proliferation, may beconveniently evaluated by cell counts, ³H-thymidine uptake and/or MTTassays.

Cytokine expression may be measured directly by capture or sandwichenzyme linked immunosorbent assays (ELISA), or indirectly by cell growthassays in which the cytokine of interest acts as a growth factor orinhibitor. Similarly, cytokine expression may be evaluated bydetermining the level of expression of mRNA coding for the cytokine byemploying reverse transcriptase polymerase chain reaction (RT-PCR) or byin-situ hybridization protocols utilizing single cells and specificoligonucleotides probes as is known in the art.

The protective immune response generated by the vaccine will typicallybe predominantly mediated by Th1 T-lymphocytes which differentiate fromproliferating CD4⁺ T-lymphocytes in the presence of IL-12 and IFN-γ.IL-12 is produced by antigen presenting cells in the early stages ofactivation. Th1 T-lymphocytes stimulate infected macrophages throughsecretion of IFN-γ and interaction of the CD40 ligand expressed by Th1cells with the CD40 receptor expressed by macrophages. More broadly, Th1cells stimulate the antibacterial mechanisms of phagocytic cells (eg.neutrophils and macrophages) and release cytokines that attract suchphagocytic cells to sites of infection. Besides IFN-γ, Th1 cellstypically also secrete IL-12 and TNF-β.

While both Th1 and Th2 cells secrete IL-3, GM-CSF and for instanceTNF-α, the overall cytokine profiles for Th1 and Th2 cells aredifferent. More particularly, activation of Th2 cells resultspredominantly in a humoral immune response characterized by theactivation of B-lymphocytes and the generation of antibodies by theactivated B cells, while Th1 cells mediate a predominantly non-antibodycellular immune response. Cytokines characteristic of Th2 cell drivenimmune response include IL-4, IL-5, IL-10, IL-13 and TGF-β. Hence,detection of the secretion of one or more of IL-12, IFN-γ, or othercytokines characteristic of activated antigen-presenting cells and Th1committed CD4⁺ T-lymphocytes, is useful in evaluating the capacity of agiven microbial isolate to activate the common mucosal system.Preferably, the level of IL-12 secretion will be measured to provide anindication of the degree of activation of antigen presenting cells bythe microbial isolate(s) being evaluated. Similarly, the level of IFN-γsecretion will typically be measured to provide an indication of thelevel of T-lymphocytes activation achieved by the microbial isolate(s).

In particularly preferred embodiments, the activation data may comprisea ratio indicating expression IL-10 relative to IL-12. IL-10 inhibitsthe release of cytokines such as IL-12 by macrophages and so inhibitsTh1 cell activation. The ratio is therefore indicative of the level of aTh1 lymphocyte response elicited by a microbial isolate. Thus, anisolate selected for activating the antigen responsive cells willdesirably elicit a cytokine response characterized by a low IL-10:IL-12ratio but high expression of IFN-γ.

Preferably, the ratio will be less than 30, more preferably less than20, 15, 10, 5 and even 4.

The vaccine will typically comprise the selected bacterial isolate(s) inan amount of between about 5% to about 80% w/w of the vaccinecomposition. As will be appreciated, the number of each isolate in thevaccine will be such that an effective dosage will be delivered to themammal for activation of the common mucosal system taking into accountthe proposed mode of delivery and nature of the vaccine (eg. powder,liquid, aerosol delivery etc). The dosage of the, or each, bacterialisolate administered will typically be in a range of from about 10⁹ toabout 10¹² cfu, and more preferably from about 10¹⁰ to about 10¹¹ cfu,respectively. The optimum dosage of a selected bacterial isolate can bedetermined by administering different dosages of the isolate todifferent groups of test mammals, prior to subsequently infecting theanimals in each group with the corresponding live bacterial pathogen,and determining the dosage level required to achieve satisfactoryclearance of the pathogen as would be well understood by the skilledaddressee.

The vaccine itself may be freeze-dried or lyophilized for laterreconstitution utilizing a physiologically acceptable buffer or fluid.The vaccine can also contain one or more anti-caking agents, isotonicagents, preservatives such as thimerosal, stablizers such as amino acidsand sugar moieties, sweetening agents such sucrose, lactose orsaccharin, pH modifiers sodium hydroxide, hydrochloric acid, monosodiumphosphate and/or disodium phosphate, a pharmaceutically acceptablecarrier such as physiologically saline, suitable buffers, solvents,dispersion media and isotonic preparations. Use of such ingredients andmedia for pharmaceutically active substances and vaccines is well knownin the art. Except insofar as any conventional media or agent isincompatible with the bacterial isolate(s), their use in vaccines ofinvention is specifically encompassed. Supplementary active agents suchas one or more cytokines for boosting the immune response, particularlycytokines characteristic of a Th1 response such as IFN-γ, IL-12 andTNF-β, can also be incorporated in the vaccine if desired.

In addition, a vaccine embodied by the invention may also comprise oneor more adjuvants. Suitable adjuvants, pharmaceutically acceptablecarriers and combinations of ingredients useful in vaccine compositionsof the present invention may for instance be found in handbooks andtexts well known to the skilled addressee such as “Remington” TheScience and Practice of Pharmacy (Mack Publishing Co., 1995)”, thecontents of which is incorporated herein in its entirety by reference.

The oral killed bacterial vaccine may be administered as a dry powder orin liquid form. Administration can for example be achieved by aerosolinhalation, as a dosed liquid, by instillation, or as a spray. Devicesfor facilitating for delivery of oral vaccines are well known in the artand include metered dose inhalers (MDIs), dry powder inhalers (DPIs) andnebulisers including those which use ultrasonic energy or compressed airor other propellant to achieve atomisation. Propellants which may beused in MDIs include for instance chlorofluorocarbons (CFCs) such astrichlorofluorocarbon (CFC-11) and dichlorodifluorocarbon (CFC-12) andhydrofluoroalkanes.

In order that the nature of the present invention may be more clearlyunderstood, preferred forms thereof will now be described with referenceto the following non-limiting examples.

Example 1 Variation in T-Lymphocyte Activation Following OralImmunization with Killed P. aeruginosa Oral Vaccine

A study was conducted to demonstrate the variability in the capacity ofrecipient T cells to recognize and respond to an unselected isolate ofkilled bacteria. A cohort of nine human subjects with bronchiectasiswere given two courses of killed oral P. aeruginosa (Ps.a) vaccine. Eachcourse comprised administering two tables (each tablet containing 10¹¹killed bacteria) per day for three days, with the second coursecommencing on day 28. The P. aeruginosa isolate used was not selectedother than being shown to be capable of activating the common mucosalsystem in an animal model.

Peripheral blood mononuclear cells were used as a source ofantigen-primed T cells and were isolated from heparinized blood on aFicoll/Paque density gradient. After centrifugation at 200 g for 20 minsat 4° C., the cells were collected from the plasma:ficoll interface,washed twice with PBS pH 7.4 and then resuspended at 2×20⁶ cells/mL inAIM-V medium containing penicillin (100 U/mL), streptomycin (100 μg/mL)and 2-mercaptoethanol (5×10⁻⁵M). Cells were culture in wells of a96-well flat-bottomed microtiter plate with or without Ps antigen (1μg/mL) or PHA-P (2 μg/mL) in a total volume of 200 μL of AIM-V medium.After incubation for 4 days at 37° C. in a CO₂ incubator, the cultureswere pulsed with 0.5 μCi per well of ³H-thymidine for the final 8 hrsbefore harvesting on a glass fibre filter using a cell harvester. Afterdrying, the filters were counted in a scintillation counter. The resultswere expressed as mean cpm±SE.

Table 1 shows the post oral immunization mean counts (H³-thymidineuptake representing antigen-induced proliferation) and the standarderrors (SE's) of the means (representing the variation in individualresponses).

TABLE 1 T cell Ps. a antigen induced proliferation in humans Ps.aeruginosa PHA antigen (1 ug/ml) SE (as % SE (as % DAY CPM × 00 of mean)CPM × 000 of mean) 0 84 13 3 16.5 28 82 12 4.4 65 56 80 12.5 9.5 42

The results shows that a consistent mean stimulation was inducedthroughout the study by the classical non-specific T cell mitogent PHA,with a consistent and small standard error, reflecting relativelysimilar proliferative responses. With Ps.a antigen stimulation beforeoral immunization, the standard error (SE) is of the same order as thatfound with PHA. However, at days 28 and 56 following oral immunization,a marked variation in response was noted (SE's of 64 and 42). Theseresults show considerable variation in T cell responsiveness in vivoreflecting failure to select an isolate to engage the T cell receptor ofmost of the recipients.

As a control, DA rats (four per group) were orally immunized using thesame isolate of Ps.a. Briefly, rats were given 5×10⁸ paraformaldehydekilled Pseudomonas aeruginosa in PBS daily for 5 days per week for 2weeks. Peripheral blood cells were isolated and ³H-thymidine uptakeassessed as described above. The results are shown in Table 2.

TABLE 2 T cell Ps. a antigen induced proliferation in in-bred ratsH³-thymidine SE (×000) (as % CPM (×000) of mean) Unimmunised 16 19Immunised (tablet) 35 13

A low SE of 13 was found in the in-bred DA rats (ie reflecting lowgenetic variability). It is concluded that the large SE (ie. reflectingdegree of individual variation) in the human response is mainly due tovariation in engagement of the antigen-presenting cell T-lymphocyte unitby the unselected vaccine candidate. This variation correlates withlarge differences in the level of protection observed betweenindividuals in the human study, reflected by the high level of variationin the reduction in bacterial colonization (measured by numbers ofbacteria in sputum) and in sputum purulence (measured as total whitecell count) as determined at day 31. Specifically, bacteria counts werereduced in three subjects (1, 3.0, 1.0 log), remained unchanged in fivesubjects, and increased (2 logs) in one subject. The sputum white cellcount fell with a mean of 40% reduction with a SE of 50%. Hence, theactivation of T cells (which are responsible for white cell recruitmentinto the bronchus lumen with subsequent bacterial clearance, eg. seeDunkley et al (1994) ‘A role for CD4+ T cells from orally immunized ratsin enhanced clearance of P. aeruginosa from the lung. Immunol 83,362-369) is also variable, indicating that the killed bacterial isolateutilized is not an optimal isolate for use as an oral killed bacterialvaccine in the general human population.

Example 2 Selection of an Optimal Isolate for Eliciting the Release ofCytokines

S. aureus and non-typeable H. influenzae isolates obtained from normalhuman subjects were killed by treatment with 2% paraformaldehyde inphosphate buffered saline (PBS). After incubation at room temperaturefor 1 hr, the treated bacteria were exhaustively washed in PBS and thentested for viability by culturing on mannitol salt agar or horse bloodagar plates. The killed bacteria were adjusted to 2×10⁹ cfu/mL byinterpolation from a standard regression curve for Absorbance versusCFU. For use in culture stimulation, the bacteria from each isolate wereadjusted to a final concentration of 2×10⁸ CFU per ml in serum-freeAIM-V medium containing penicillin (100 U/mL), streptomycin (100 μg/ml)and 2-mercaptoethanol (2-ME, 5×10⁻⁵M).

For S. aureus isolates, groups of male BALB/c mice (n=5) were given5×10⁹ killed whole cell bacteria by intragastric administration every 2days per week for 2 weeks before challenge with live S. aureus bacteriaadministered intranasally. The dose was determined on the basis similarstudies examining variable oral dosage. Control mice (n=5) were fed PBSalone. Reduction in colonization in the nose was determined at 24-48 hrsafter challenge. For the NTHi isolates, groups of male BALB/c mice weregiven 5×10⁹ killed whole cell bacteria by the intragastric route every 2days per week for 2 weeks followed by intranasal challenge with liveNTHi bacteria. Control mice were again fed PBS alone. Reduction in NTHicolonization was determined in bronchoalveolar washings (BAL) and lunghomogenates, at 4 hrs following live challenge.

Human peripheral blood mononuclear cells (PBMNC) were separated fromheparinized blood by centrifugation on a Ficoll/Paque density gradient.After washing by centrifugation, PBMNC were cultured in AIM-V medium at2×10⁵ cells per well with 2×10⁶/ml or 2×10⁸/ml CFU of killed bacteria ina total volume of 300 uL in wells of a 96-well flat-bottomed microtitreplate. After incubation at 37° C. and 5% CO₂ in air for 3 days, theculture supernatants were collected and stored at −20° C. until assayfor cytokines by ELISA.

The capacity of individual S. aureus and NTHi isolates to stimulatecytokine production was tested using an antigen-presenting cell-T cellculture system. Briefly, PBMNC (2×10⁶/ml) containing APC and T cellswere stimulated with graded doses of killed bacteria in flat-bottomedwells of a 96-well plate for 3 days at 37° C. in a CO₂ incubator. Theculture supernatants were collected and assayed for IL-12 and IFN-γ byELISA.

Table 3 shows the results of S. aureus isolates (A2, A3, A8, A15, A28,A19, A20) tested for their capacity to stimulate the production of IL-12and IFN-γ. The results are medians normalized and expressed as apercentage of control. Variable stimulation of IL-12 and IFN-γ wasobserved. Isolates A2, A8, A28 and A19 were greater stimulators of bothIL-12 and IFN-γ production than isolates A15, A20 and A3. Compared withthe other isolates, stimulation with isolate A28 led to a 4-10 foldproduction of IL-12 and/or IFN-γ.

TABLE 3 Production of IL-12 and IFN-γ in cultures of peripheral bloodmononuclear cells stimulated with different doses of killed whole cellS. aureus isolate. IL-12 pg/mL IFN-γ pg/mL Isolates *10⁶ bacteria/l *10⁸bacteria/l 10⁶ bacteria/l 10⁸ bacteria/l A2 147 186.5 2159 891.9 A3 <10<10 2242 2009 A8 36.5 119 1938 1711 A15 <10 <10 <10 2476 A28 1568 <108592 9202 A19 149.3 267.8 800.6 607.4 A20 18.6 26.7 977.9 668.3 *numbersof killed bacteria per ml

When isolates A2, A8 and A28 were tested in cultures using PBMNC from 5normal healthy subjects, only isolates A2 and A28 were capable ofstimulating substantial amounts of IL-12 and IFN-γ in all subjects(Table 4 and Table 5). These results indicate that isolates A2 and A28are potent stimulators of immunomodulating cytokines and, therefore,suitable as candidate immunostimulators, likely to mediate protectivemucosal immunity in most recipients.

TABLE 4 Production of IL-12 in cultures of human peripheral bloodmononuclear cells from normal healthy subjects stimulated with killedwhole cell S. aureus isolate IL-12 (pg/mL) Iso- Subject 1 Subject 2Subject 3 Subject 3 Subject 5 lates *10⁶ *10⁸ 10⁶ 10⁸ 10⁶ 10⁸ 10⁶ 10⁸10⁶ 10⁸ A2 138.6 193.7 11.3 147.5 <10 87.6 nd 264.8 nd 271.8 A8 157 <1046 10.6 27.6 29.7 nd nd nd nd A28 112 214.2 <10 137.2 nd 56.9 nd 328.2nd 454.2 None — <10 — <10 — <10 — <10 — <10 *number of killed bacteriaper mL nd = not determined

TABLE 5 IFN-γ production stimulated with killed whole cell S aureusisolate in cultures of human peripheral blood mononuclear cells fromnormal healthy subjects IFN-γ (pg/mL) Subject 1 Subject 2 Subject 3Subject 3 Subject 5 Isolates *10⁶ *10⁸ 10⁶ 10⁸ 10⁶ 10⁸ 10⁶ 10⁸ 10⁶ 10⁸A2 1745 5501 3316 8689 963 8838 nd 12152 nd 6232 A8 3388 4628 7458 74586479 8285 nd 11770 Nd nd A28 724 5368 8547 1032 646 8429 nd 13560 Nd6544 None — <10 — <10 — <10 — <10 — <10 *numbers of killed bacteria perml nd = not determined

All NTHi isolates tested (B3, B4, B6, B10 and B14) were found capable ofstimulating production of IL-12 and IFN-γ in variable amountsirrespective of the doses of bacteria used in culture stimulation (Table6). Isolates B3 and B6 stimulated the generation of higher amounts ofIL-12 and IFN-γ compared to the other NTHi isolates. The results aremedians expressed as a percentage of control.

TABLE 6 Production of IL-12 and IFN-γ in cultures of peripheral bloodmononuclear cells (PBMNC) stimulated with killed whole cell non-typableH. influenzae isolate IL-12 (pg/mL) IFN-γ (pg/mL) Bacterial 10⁶ bact./ml10⁸ bact./ml 10⁶ bact./ml 10⁸ bact./ml Isolates *PC *RC PC RC PC RC PCRC B3 65.5 20 42.8 20.6 1740 316 1723 786 B4 25 13.2 18.2 14.8 956 761208 420 B6 40.5 26.9 41.2 34.7 1755 436 2063 1758 B10 18.2 16.2 24.421.2 378 53 1002 619 B14 28.6 17.1 18.2 16.8 274 45 1341 713 None 23 — —— 19 — — — *PBMNC from normal healthy subjects

Example 3 Selection of an Optimal Isolate of Bacteria as anImmunostimulator to Reduce Mucosal Colonization

Two mouse mucosal colonization models were used to determine (1) thereduction in colonization of S. aureus in the nasal cavities and (2) thereduction in colonization of non-typeable H. influenzae in the lungsfollowing challenge with live bacteria.

In model 1, 8 week old male SPF BALB/c mice (n=5) were given killedwhole cell S. aureus bacteria (5×10⁹ per mouse) in 300 ul PBS by theintragastric route every 2 days per week for 2 weeks. Control micereceived PBS alone. Paraformaldehyde-killed bacteria from isolates A2,A28, A3, A15, A19 and A20 were tested including an ATCC strain as areference (ATCC 49247), respectively. One day after the final dose, micewere challenged with live S. aureus by administering 10 μl of 5×10⁹bacteria/ml into each nostril. At 24-48 hrs after challenge, mice weresacrificed and the nasal tissues excised. The tissues were homogenizedin PBS and the homogenates collected then assayed for bacteriaconcentration by plating 10-fold serial dilutions of the homogenates onmannitol salt agar plates. CFU counts were determined after 24 hrincubation at 37° C.

FIG. 2 shows that mice receiving killed bacteria prepared from isolatesA19, A20, A28, A2, A3, A8 and A15 had reduced in compared with controls.The levels of protection were dependent on the isolates chosen witheffectiveness being A28>A2>A8>A3>A15>A20>A19. Furthermore, the highlevels of protection achieved with A28, A2 and A8 isolates correlatedwith their capacity to stimulate high levels of IL-12 and IFN-γ incultures of human PBMNC (see Tables 3 and 4).

In model 2, mice (n=5) were given killed whole cell NTHi (5×10⁹ permouse) in 300 ul of PBS by the intragastric route every 2 days per weekfor 2 weeks before challenge with 10 ul of live bacteria in PBS (5×10⁹per ml) administered by the intranasal route. Paraformaldehye-killedbacteria prepared from isolates B3, B14, B16 and B10 were tested,respectively. At 4 hrs following live challenge, the reduction incolonization in terms of bacterial clearance was determined inbroncho-alveolar lavage fluid (BAL) and lung homogenates from treatedand control mice. As shown in FIG. 1, of the isolates tested, B3, B16and B10 gave the best clearance rate compared to the ATCC referencestrain and isolate B14. When compared for in vitro stimulation of IL-12and IFN-γ production, B3 and B6 isolates gave the best correlation withreduction in colonization (see Table 4).

Example 4 Selection of an Optimal Isolate of Bacteria as a CandidateVaccine on the Basis of Optimal Cytokine Response In Vitro andProtection In Vivo

Peripheral blood mononuclear cells (PBMNC) were separated fromheparinized blood from normal healthy subjects by centrifugation onFicoll/Paque density solution. The cells were collected from theplasma/ficoll interface and washed three times in PBS by centrifugationat 200 g for 15 mins at 4° C. Before culture, the cells werere-suspended in AIM-V serum-free medium at 2×10⁶ cells/mL and theviability determined by trypan-blue exclusion test. One hundredmicroliter aliquots of the cell suspension were added in duplicates toequal volumes of AIM-V medium containing graded concentrations of killedbacteria or medium alone in wells of a flat-bottomed 96-well microtitreplate. The cultures were incubated for 3 days at 37° C. in an incubatorwith 5% CO₂ in air. After incubation, the supernatants were collected,pooled and stored at −20° C. until assay.

IL-10, IL-12 and IFN-γ in culture supernatants were measured by ELISAusing monoclonal antibody pairs with recombinant proteins as standards.Briefly, wells of a flat-bottomed microtiter plate were coated withcapture antibody overnight at 4° C. After washing, the wells wereblocked with 2% BSA solution in PBS for 60 mins at room temperature (RT)and followed by washing in PBS/Tween. One hundred microliters of samplesupernatant was added to each well. Following incubation for 90 mins atRT, the wells were washed and then 100 ul of biotinylated detectorantibody was added. After incubation for 90 mins, HRP-conjugatedstreptavidin solution was added for 30 mins. The wells were then washedbefore adding TMB substrate solution for 20 mins. After colourdevelopment, the reaction was stopped with sulphuric solution beforereading in an ELISA plate reader at 450 nm. The amount of cytokinesecreted in culture supernatant was calculated by interpolation usingthe standard curve and the results were expressed as pg/mL.

Table 7 shows that high levels of IFN-γ and IL-12 produced by peripheralblood mononuclear cells stimulated with S. aureus (isolate A28) or NTHi(isolate B6) correlated with high levels of protection in the mousemodel of colonization. In contrast, there was no correlation betweenisolates (A3 and B10) which stimulated low levels of cytokines and highlevels of protection. Conversely, no correlation was observed betweenisolates (A19 and B14) which stimulated high levels of cytokines and lowlevels of protection. The results demonstrate that optimal cytokineresponse and in vivo protection are markers for the selection of anoptimal isolate of bacteria as a candidate oral vaccine orimmunostimulator to enhance mucosal immunity and reduce colonization inthe airways.

TABLE 7 Correlation of in vitro cytokine production with in vivobacterial clearance. Isolate IL-12 (pg/mL) IFN γ (pg/mL) Protection (%)A28 1568 9202 91.3 A3 <10 <10 74.7 A19 267.8 800.6 28 B6 412 1063 100B10 <10 335 72.2 B14 24 1341 24

Example 5 Selection of an Optimal Isolate Based on DifferentialProduction of IL-10 and IL-12, and the Capacity to Enhance IFN-γProduction and Protection In Vivo

Stimulation of PBMNC in culture by different isolates produced differentamounts of IL-10 and IL-12 whose ratios correlated with the levels ofIFN-γ and in vivo protection. As shown in Table 8, NTHi isolates (B3,B6) with a low IL-10/IL-12 ratio were better stimulators of a Th1response characterized by maximal production of IFN-γ and decreasedcolonization of bacteria in the airways of mice orally immunized withkilled vaccine isolates compared with controls. Conversely, isolates(B4, B10 and B14) with high IL-10/IL-12 ratios correlated with lowerproduction of IFN-γ and higher levels of colonization. This was alsotrue for S. aureus isolates where high levels of IFN-γ correlated withlow IL-10/IL-12 ratios and low rates of colonization (Table 9). Takentogether, the results show that an optimal isolate of bacteria can beselected as a candidate vaccine on the basis of low IL-10/IL-12 ratioand high IFN-γ and an enhanced protection in vivo. Thus, on the basis ofthese results, an optimal isolate would be associated with a cut-offvalue of IL-10/IL-12 ratio of say <15 and high production of IFN-γ (786pg/ml for NTHi and >500 pg/ml for S. aureus) for all healthy subjectstested (minimum of 3 subjects).

TABLE 8 Selection of non-typable H influenzae isolates based onIL-10/IL-12 ratio and IFN-γ production and protection in vivo Subject 1Subject 2 Subject 3 IL-10/ IL-10/ IL-10/ Bacteria NTHi IL-12 IFN-γ IL-12IFN-γ IL-12 IFN-γ Colonisation* Isolate Ratio (pg/ml) Ratio (pg/ml)Ratio (pg/ml) % of Control B3 10.9 1010 2.0 1740 1.25 786 37 B6 7 16452.5 2063 0.41 1758 55 B4 23.8 532 26.4 956 6.7 76 100 B10 720 236 16.8690 80 53 100 B14 749 45 22 897 29.8 45 76 Control 51 0 10 23 1.24 19100 *Colonisation rate in the airways of mice (n = 10) orally immunisedwith killed whole cell bacteria isolate or mice given PBS only (Control)

TABLE 9 Selection of S. aureus isolates based on IL-10/IL-12 ratio andIFN-γ production and protection in vivo Subject 1 Subject 2 Subject 3IL-10/ IL-10/ IL-10/ Bacteria S. aureus IL-12 IFN-γ IL-12 IFN-γ IL-12IFN-γ Colonisation* Isolate Ratio (pg/ml) Ratio (pg/ml) Ratio (pg/ml) %of Control A2 0.99 5501 2.5 8639 5.9 8839 9.7 A8 3.75 4623 13.2 762010.3 8250 26.2 A28 1.6 5638 4.9 8547 14.3 8429 8.7 A19 24 197 3.9 60720.9 714 72 A20 18 222 38.8 668 125.3 1075 62.5 Control 2 9.8 2 198 2.3230 100 *Colonisation rate in the noses of mice (n = 10) orallyimmunised with killed whole cell bacteria isolate or mice given PBS only(Control)

Example 6 Stimulation of Peripheral Blood Mononuclear Cells byNon-Typeable H. influenzae Isolate NTHi 164

Whole killed NTHi 164 was cultured with PBMNC from normal healthycontrols and IFN-γ production assessed. The results are shown in Table10.

TABLE 10 Stimulation of IFN-γ production in PBMNC stimulated with wholekilled NTHi 164 isolate Subject Whole killed MD PH PC RC NTHi 164 IFN-γ(pg/ml) 10⁶ bacteria/mL 207 257 491 72 10⁸ bacteria/mL 1738 1193 4533514

Compared to Table 6 in Example 2, NTHi 164 expressed comparable levelsof IFNγ for RC and higher level for PC than elicited by NTHi isolates B3or B6. A particulate fraction of the NTHi isolate was also prepared andthe capacity of the fraction to stimulate IFN-γ production assessed. Theresults are shown in Table 11. Briefly, NTHi 164 was revived from −80°C. storage onto chocolate agar plates then subcultured on chocolateagar. The bacteria were harvested, pelleted, washed and resuspended to1010/ml in PBS. The suspension was sonicated at an amplitude of 6μ using10 cycles of 30 seconds on and 60 seconds off. The suspension wascentrifuged at 7000 g and the supernatant collected, sterile filtered,and protein content determined by Pierce BCA protein assay. Aliquotswere stored frozen at −20° C. until use.

TABLE 11 Stimulation of IFN-γ production in PBMNC particulate antigen ofwhole killed NTHi 164 isolate Subject NTHi 164 1 2 3 4 5 6 7 antigenIFN-γ (pg/ml)  1 μg/mL 130 287 24 111 156 86 35 10 μg/mL 368 128 101 169356 565 334

Tables 10 and 11 show that both whole killed NTHi 164 and theparticulate antigen extract of the isolate stimulates peripheral bloodmononuclear cells to produce IFN-γ. The production of IFN-γ is IL-12dependent.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

REFERENCES

-   1. Dunkley et al, Immunol. (1994) 83, Pp. 362-369-   2. Remington. “The Science and Practice of Pharmacy.” (Mack    Publishing Co., 1995)

1. A method for selecting at least one microbial isolate for use in anoral killed vaccine against abnormal microbial colonization of a mucosalsurface, of the selected microbial isolate or isolates, the methodcomprising: evaluating capacity of a plurality of different isolates ofa unicellular microbe to activate antigen responsive cells to provideactivation data for each microbial isolate; evaluating effectiveness ofthe isolates in reducing infection of a mucosal surface by the microbeto provide clearance data for each microbial isolate; and selecting atleast one isolate from the microbial isolates, the activation data andclearance data for which correlate and is optimal for generating mucosalimmunity against the microbe compared to the, or each, other of theisolates, or an isolate the activation data for which is optimal and afurther isolate the clearance data for which is optimal, compared tothe, or each, other of the isolates, respectively, for use informulating the vaccine.
 2. A method according to claim 1 wherein themicrobial isolates are killed microbial isolates.
 3. A method accordingto claim 1 wherein an isolate for which both the activation data and theclearance data is optimal compared to the other of the isolates isselected.
 4. A method according to claim 1 wherein an isolate for whichthe activation data is optimal and a further isolate for which theclearance data is optimal, compared to the, or each, other of theisolates, are selected.
 5. A method according to claim 1 wherein themucosal immunity predominantly comprises a cellular immune response. 6.A method according to claim 1 wherein the activation data comprisesantigen presenting cell data indicative of capacity to activate antigenpresenting cells and data indicative of capacity to activateT-lymphocytes, and wherein the antigen presenting cell data and theT-lymphocyte activation data correlate.
 7. A method according to claim 6wherein the antigen presenting cell data comprises data indicative ofcapacity to induce IL-12 expression by the antigen responsive cells. 8.A method according to claim 7 wherein the antigen presenting cell datacomprises data indicative of IL-12 expression relative to expression ofIL-10 by the antigen responsive cells.
 9. A method according to claim 8wherein an isolate the activation data for which is indicative ofcapacity to elicit a cytokine response characterized by an IL-10:IL-12ratio of about 30 or less is selected for use in the vaccine.
 10. Amethod according to claim 6 wherein the further data comprises dataindicative of capacity to induce IFN-γ expression by the antigenresponsive cells.
 11. A method according to claim 1 wherein the microbeis selected from the group consisting of bacteria, yeast or fungi.
 12. Amethod according to claim 11 wherein the microbe is selected frommicrobial species selected from the group consisting of Chlamydiaspecies, Haemophilus species, Non-typeable Haemophilus species,Pseudomonas species, Streptococcus species, Staphylococcus species, E.coli species, Mycoplasma species, Helicobacter species, Candida speciesand Saccharomyces species.
 13. A method according to claim 12 whereinthe microbe is selected from Non-typeable H. influenzae, S. aureus, P.aeruginosa and S. pneumoniae.
 14. A method for providing an oral killedvaccine against abnormal microbial colonization of a mucosal surface, ofat least one selected microbial isolate the method comprising:evaluating capacity of a plurality of different isolates of aunicellular microbe to activate antigen responsive cells to provideactivation data for each microbial isolate; evaluating effectiveness ofthe isolates in reducing infection of a mucosal surface by the microbeto provide clearance data for each microbial isolate; selecting at leastone isolate from the plurality of isolates, the activation data andclearance data for which correlate and is optimal for generating mucosalimmunity against the microbe compared to the, or each, other of theisolates, or an isolate the activation data for which is optimal and afurther isolate the clearance data for which is optimal, compared tothe, or each, other of the microbial isolates, respectively, for use informulating the vaccine; and wherein the vaccine is formulated by mixingthe selected isolate or isolates with one or more pharmaceuticallyacceptable excipients suitable for oral administration.
 15. A methodaccording to claim 14 wherein the microbial isolates are killedmicrobial isolates.
 16. A method according to claim 14 wherein anisolate for which both the activation data and the clearance data isoptimal compared to the, or each, other of the isolates is selected. 17.A method according to claim 14 wherein an isolate for which theactivation data is optimal and a further isolate for which the clearancedata is optimal, compared to the, or each, other of the isolates, areselected.
 18. A method according to claim 14 wherein the mucosalimmunity predominantly comprises a cellular immune response.
 19. Amethod according to claim 14 wherein the antigen responsive cellscomprise one or more cell types selected from the group consisting ofantigen presenting cells and T-lymphocytes.
 20. A method according toclaim 19 wherein the antigen responsive cells comprise both antigenpresenting cells and T-lymphocytes.
 21. A method according to claim 20wherein the antigen presenting cells comprise macrophages.
 22. A methodaccording to claim 20 wherein the T-lymphocytes are Th1 lymphocytes orTh1 committed T-lymphocytes.
 23. A method according claim 14 wherein theactivation data comprises antigen presenting cell data indicative ofcapacity to activate antigen presenting cells and data indicative ofcapacity to activate T-lymphocytes, and wherein the antigen presentingcell data and the T-lymphocyte activation data correlate.
 24. A methodaccording to claim 23 wherein the antigen presenting cell data comprisesdata indicative of capacity to induce IL-12 expression by the antigenresponsive cells.
 25. A method according to claim 24 wherein the antigenpresenting cell data comprises data indicative of IL-12 expressionrelative to expression of IL-10 by the antigen responsive cells.
 26. Amethod according to claim 25 wherein an isolate the activation data forwhich is indicative of capacity to elicit a cytokine responsecharacterized by an IL-10:IL-12 ratio of about 30 or less is selectedfor use in the vaccine.
 27. A method according to claim 23 wherein theT-lymphocyte activation data comprises data indicative of capacity toinduce IFN-γ expression by the antigen responsive cells.
 28. A methodaccording to claim 14 wherein the effectiveness of the isolates toreduce infection of the mucosal surface is evaluated by determiningcapacity of the isolates to reduce the infection of the mucosal surfacein vivo.
 29. A method according to claim 14 wherein the microbe isselected from the group consisting of bacteria, yeast or fungi.
 30. Amethod according to claim 29 wherein the microbe is selected frommicrobial species selected from the group consisting of Chlamydiaspecies, Haemophilus species, Non-typeable Haemophilus species,Pseudomonas species, Streptococcus species, Staphylococcus species, E.coli species, Mycoplasma species, Helicobacter species, Candida speciesand Saccharomyces species.
 31. A method according to claim 30 whereinthe microbe is selected from Non-typeable H. influenzae, S. aureus, P.aeruginosa and S. pneumoniae.
 32. A method for providing an oral killedvaccine against abnormal microbial colonization of a mucosal surface, ofat least one selected microbial isolate the method comprising:evaluating capacity of a plurality of different isolates of aunicellular microbe to induce expression of IL-10 and IL-12 in antigenresponsive cells; selecting at least one isolate from the microbialisolates, that induces optimal expression of IL-12 relative to IL-10compared to the, or each, other of the isolates, respectively, for usein formulating the vaccine; and wherein the vaccine is formulated bymixing the selected isolate or isolates with one or morepharmaceutically acceptable excipients suitable for oral administration.33. A method according to claim 32 wherein the selected isolate inducesexpression of the IL-10 and IL-12 at a ratio of IL-10 to IL-12 of about30 or less.
 34. A method according to claim 33 wherein the ratio isabout 15 or less.
 35. A method according to claim 32 wherein the isolatefurther induces expression of IFN-γ by the antigen responsive cells. 36.A method according to claim 35 wherein the expression of IFN-γ by theisolate is optimal compared to the, or each, other of the isolates,respectively.
 37. A method according to claim 32 wherein the antigenresponsive cells comprise peripheral blood mononuclear cells.
 38. Amethod according to claim 32 wherein the antigen responsive cellscomprise antigen presenting cells and T-lymphocytes.
 39. A methodaccording to claim 32 wherein the microbe is selected from microbialspecies selected from the group consisting of Chlamydia species,Haemophilus species, Non-typeable Haemophilus species, Pseudomonasspecies, Streptococcus species, Staphylococcus species, E. coli species,Mycoplasma species, Helicobacter species, Candida species andSaccharomyces species.
 40. A method according to claim 39 wherein themicrobe is selected from Non-typeable H. influenzae, S. aureus, P.aeruginosa and S. pneumoniae.
 41. An oral killed vaccine prepared by themethod of claim
 14. 42. An oral killed vaccine prepared by the method ofclaim 32.